1. Field of the Invention
The present invention relates to a method for the separation of polysaccharides. More particularly, the present invention relates to a method for the separation of polysaccharides using conjugates of thermo-sensitive polymers and ligands.
2. Description of the Related Arts
Carbohydrate-containing molecules play important roles in many aspects of-human physiological responses, including the modulation of the immune system and exertion of tumor-inhibitory effects. At the molecular level, cell-cell recognition as well as signal transduction has been noted to involve carbohydrate-protein interactions.
In addition to their role as nutrient for energy production, carbohydrate-containing molecules are also believed to participate in important regulatory and functional roles in metabolism. Moreover, some polysaccharides are considered as immunonutrients; for example, compounds extracted from several mushrooms, in particular (1xe2x86x923)-xcex2-D-glucans, show the ability to modulate the immune system as well as anti-inflammatory responses and exhibit potential antitumor activity (Borchers AT, et al. Mushrooms, tumors, and immunity. Proc Soc Exp Biol Med, 1999, 221:281-93). Polysaccharides prepared from many species of fungi such as Cordyceps sinensis and Ganoderma lucidum (Ling Zhi mushroom) are now widely used as health food supplements in Asia and have been shown to provide effects beneficial to general health; however, the mechanisms involved are poorly understood. An understanding of the molecular mechanisms involved requires an effective separation method, providing sufficient purity while preserving activity.
To elucidate the mechanisms of these glycoproteins involved in the various biological processes, one needs to embark on an arduous task of first separating the polysaccharide in reasonably purity from their indigenous biological mixtures. In general, it is necessary to conduct these separations at a temperature which does not inhibit biological activity due to heat denaturation. Lastly, the chemical heterogeneity of the polysaccharides involved may complicate the understanding of the underlying mechanisms. Because of these factors, the study of glycobiology has fallen behind those of nucleotides and proteins.
xe2x80x9cIntelligent (smart) polymersxe2x80x9d, or so called xe2x80x9cstimuli-responsive polymersxe2x80x9d or xe2x80x9cenvironmentally sensitive polymersxe2x80x9d, show distinct property changes to small changes in environmental conditions, such as solution pH, ionic strength, solvent composition, temperature, light, and electric field. These polymers have applied in medicine and biotechnology. A few examples are cell and protein attachment/detachment used in therapeutic devices, bioactive surfaces for enzyme immobilization, drug delivery and affinity separation (Hoffman AS, et al. Conjugation of stimuli-responsive polymers and biomolecules: random and site-specific conjugates of temperature-sensitive polymers and proteins. Macromol Symp, 1997; 118:553-563).
Purification using these polymers on biomolecules usually involves the attachment of biotin to polymers such as poly(N-isopropylacrylamide). This conjugate is then used to separate streptavidin-containing mixtures, as in U.S. Pat. No. 5,998,588. As best as we know, there have not been any efforts in attaching affinity ligands to these polymer networks for the purification of polysaccharides and related compounds.
It is therefore a primary object of the present invention to develop a rapid, technically simple and cost effective method for the purification/partial purification of bioactive polysaccharides by introducing a smart polymer in the affinity purification.
The method for the separation of polysaccharides in the invention comprises conjugating a ligand to a thermo-sensitive polymer to form a conjugate, wherein the ligand specifically recognizes a polysaccharide, and the thermo-sensitive polymer features a lower critical solution temperature (LCST), contacting a mixture containing a desired polysaccharide with the conjugate at an environmental temperature lower than LCST to form a polysaccharide-conjugate complex, collecting the polysaccharide-conjugate complex at an environmental temperature higher than LCST, and releasing the polysaccharide from the polysaccharide-conjugate complex to obtain the desired polysaccharide.